Vitis Vinifera Leaf Extract Protects Against Glutamate-Induced Oxidative Toxicity in HT22 Hippocampal Neuronal Cells and Increases Stress Resistance Properties in Caenorhabditis Elegans

Vitis vinifea has been used for traditional medicines, food, beverages, and dietary antioxidant supplements. The chemical compositions and biological activities of the fruits and seeds have been extensively investigated. However, the biological effects of the leaves are limited, and its anti-neurodegeneration or antiaging activities are little known. The current work aims to study the beneficial effects of V. vinifera leaf extract on neuroprotective effects in HT22 cells, antiaging, and oxidative stress resistance properties in the Caenorhabditis elegans model. The ethanol extract was characterized by phytochemical composition using gas/liquid chromatography–mass spectrometry and reversed-phase high-performance liquid chromatography. The beneficial effects of V. vinifera ethanol (VVE) extract on antioxidant properties, neuroprotective effects, and the underlying mechanisms were studied by in vitro and in vivo studies. In HT22 cells, we found that VVE has a protective effect against glutamate-mediated oxidative stress-induced cell death. The gene expression of cellular antioxidant enzymes such as CAT, SODs, GSTs, and GPx was upregulated by VVE treatment. Moreover, VVE was also shown to alleviate oxidative stress and attenuate reactive oxygen species accumulation in C. elegans. We demonstrated that VVE could upregulate the expression of stress-response genes gst-4 and sod-3 and downregulate the expression of hsp-16.2. Our results suggest that the oxidative stress resistance properties of VVE are possibly involved in DAF-16/FoxO transcription factors. VVE reduced age-related markers (lipofuscin) while did not extend the life span of C. elegans under normal conditions. This study reports the neuroprotective effect and antioxidant activity of V. vinifera leaf extract and suggests its potential as a dietary or alternative supplement to defend against oxidative stress and age-related diseases.

Vitis vinifera L. (grape) has been used for food, beverages, and traditional medicine. The leaves have been used in hemorrhoid and diabetic treatments (5). The therapeutic effects are mainly attributed to the phenolic compounds in the fruits, including flavonoids, anthocyanins, and proanthocyanidins (6). These compounds have an antioxidant capacity and antibiotic, antiallergic, antidiarrhea, antiulcer, and anti-inflammatory effects (7,8). Evidence suggests that the grape seed and skin extracts have a lifespan-extending effect in C. elegans (9). The leaf extract of V. vinifera has antioxidant and anti-inflammatory activities (10). However, the neuroprotective effects and oxidative stress resistance properties of V. vinifera leaf extract in C. elegans have not been reported.
In the current study, the neuroprotective effects of V. vinifera leaf extract against glutamate-induced cytotoxicity in HT22 cells, oxidative stress resistance properties, and antiaging in C. elegans were investigated. This study reports novel neuroprotective effects and antioxidant activity of the V. vinifera leaf extract and suggests novel dietary supplements to defend against oxidative stress and age-associated neurodegenerative diseases.

Plant Material and Extraction
The leaves of V. vinifera were collected from the Pak Chong district, Nakhon Ratchasima Province, Thailand (14.7125 • N, 101.421944 • E) in July 2016. A voucher specimen of V. vinifera (BCU-016295) has been deposited at the herbarium of Kasin Suvatabhandhu, Department of Botany, Faculty of Science, Chulalongkorn University, Thailand.
The leaves of V. vinifera were dried at shadow for 1-2 weeks and were grounded into a powder. The powder sample (40 g) was subjected to sequential extraction with solvents of different polarities (hexane, dichloromethane, and ethanol at boiling temperature 70-80 • C) by Soxhlet for 36 h (11,12). The supernatants were combined, subsequently filtered (Whatman No. 1 filter paper), and evaporated at 35-45 • C by using a vacuum evaporator. The crude extracts were stored at −20 • C as a stock. The residue was dissolved in DMSO to a final concentration of 100 mg/ml as a stock solution before the experiments.
The extraction yields of hexane, dichloromethane, and ethanol fractions were 1.32, 0.35, and 18.93%, respectively. The therapeutic effects of V.vinifera products are mainly attributed to the phenolic compounds (6). Ethanol has been frequently used as a solvent for polyphenol extraction and is safe for human consumption (13). Moreover, the ethanol fraction showed the highest yield compared with hexane and dichloromethane fractions. Thus, the V. vinifera ethanol extract was used in this study.

Qualitative Phytochemical Screening
The phytochemical composition of the ethanol extract was analyzed using gas/liquid chromatography-mass spectrometry (LC-MS) (

Radical Scavenging Activity
The antioxidant activity of the V. vinifera ethanol (VVE) extract was determined by measuring the decrease in the absorbance of the stable free radical ABTS and DPPH, following our methods as described previously (15). Briefly, the DPPH and ABTS were prepared in ethanol at 0.2 mg/ml. The reaction consisted of ABTS or DPPH solution and different concentrations of the VVE extract at a 9:1 ratio. The mixture was incubated in the dark for 30 min at RT. The absorbance values of DPPH and ABTS were measured at 734 nm or 517 nm, respectively, using an EnSpire R Multimode Plate Reader (Perkin-Elmer). The percent inhibition values of the radical and IC50 were calculated as described previously (15). The antioxidant capacity was expressed as vitamin C equivalent antioxidant capacity in milligrams per gram of dry weight plant extract (15).

Total Phenolic Content
The assay was carried out according to the Folin-Ciocalteu method and described in our previous work (15). In brief, a Folin-Ciocalteu's phenol reagent (10-fold diluted) and the extract (1 mg/ml) were mixed in a 1:1 ratio and incubated for 20 min. Next, a 7.5% (w/v) Sodium carbonate solution was added to the mixture and kept in the dark at RT for 20 min. The absorbance was read at 760 nm using an EnSpire R Multimode Plate Reader (Perkin-Elmer) as described previously (15). The calibration curve of standard (gallic acid) was used to calculate the total phenolic content, expressed as gallic acid equivalents (GAE.g of plant extracts).

Total Flavonoid Content
The assay was carried out according to the aluminum chloride colorimetric method and described in our previous work (15). Briefly, the extract was mixed with 10% (v/v) aluminum chloride solution and 1-M sodium acetate solution, followed by incubating for 40 min in the dark. After that, the absorbance was measured at 415 nm. The calibration curve of standard (quercetin) was used to calculate the total flavonoid content, expressed as quercetin equivalents (QE.g of plant extracts).

Cell Culture
Mouse hippocampal HT22 cells were obtained from Professor David Schubert (Salk Institute, San Diego, CA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under 5% carbon dioxide at 37 • C.

Cell Treatment
HT22 cells were seeded in tissue culture plates (5,000 cells/well in 96 well-plates, 8,000-10,000 cells/well in 12 well-plates) for 12-18 h. After that, cells were treated with different concentrations of VVE extract (10-100 µg/ml) for 48 h. To induce 40-50% cell toxicity, the culture medium was added with 5-mM glutamate and incubated for 18 h. Stock solutions of glutamate and VVE extract were prepared in DMEM and DMSO, respectively. For the control group, cells were treated with 0.1% (v/v) DMSO.

Determination of Cell Viability
Cell viability was evaluated by using MTT and LDH assay (Supplementary Materials).

RNA Isolation and Quantitative Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted using the Trizol reagent (Invitrogen) following the manufacturer's instructions. Reverse transcription was done according to the recommended manufacturers' protocols of AccuPower RT PreMix (Bioneer). The q-PCR was performed in an Exicycler TM 96 (Bioneer). The PCR results were measured using fluorescent signals. The PCR conditions were: 95 • C for 15 min, denaturation at 95 • C for 15 s for 45-55 cycles, and primer annealing/extension at 55 • C for 30 s. The primer specificity test was performed by melting curve. β-actin (internal control gene) was used to normalize the relative expression levels by using the 2 − CT method. The gene-specific sequences of primers were CAT, SOD1, GPx, GSTo1, GSTa2, and β-actin (3) (Supplementary Materials).

C. elegans Strains and Culture Conditions
The strains N2 (wild type), TJ375 [gpIs1(hsp-16-2:: , CL2166 [(pAF15)gst-4p::GFP::NLS], and Escherichia coli OP50 were obtained from the Caenorhabditis Genetics Center at the University of Minnesota, USA. All strains were maintained at 20 • C and cultured on nematode growth media (NGM) plates with living E. coli OP50. For all assays, the larvae (L1 stage) were seeded in liquid medium (S-medium), inoculated with E. coli OP50. Synchronous populations were obtained by using hypochlorite treatment (5-M sodium hydroxide and 5% sodium hypochlorite). The eggs were allowed to hatch in M9 buffer as described previously (15,16). For the treatment groups, worms were treated with different concentrations of VVE extract: 25, 50, and 100 µg/ml. For the control group, worms were treated with 0.1% (v/v) DMSO.

Survival Assay Under Juglone-Induced Oxidative Stress
L1 larvae of wild-type (N2) and CF1038 transgenic strains were treated with different concentrations of VVE extract in Smedium for 48 h. After treatment, worms were exposed to the pro-oxidant juglone at 80 µM for 24 h. The dead and live worms were counted by gentle touch with a platinum wire.

Measurement of Intracellular Reactive
Oxygen Species in C. elegans L1 larvae of wild-type (N2) and CF1038 transgenic strains were treated with different concentrations of VVE extract in S-medium for 48 h. After treatment, ROS production was quantified by the DCFH-DA method according to our previous work (15,17). The 50-µM DCFH-DA was added into S-medium and incubated in the dark at 20 • C for 1 h.
Worm images were examined under a fluorescent microscope (Keyence Deutschland GmbH, Neu-Isenburg, Germany) at least 30 worms per group. The relative fluorescence of the whole body was examined using ImageJ software (National Institutes of Health, Bethesda, MD). The results are presented as mean fluorescence ± SEM.

Quantification of hsp-16.2 Expression
L1 larvae of TJ375 transgenic worms, which carry hsp-16.2 promoter regions fused with a green fluorescent protein (GFP) reporter, were treated with different concentrations of VVE extract in S-medium at 20 • C for 72 h. Then, the worms were induced by exposing a nonlethal dose of 20-µM juglone for 24 h. After incubation, worms were anesthetized by the addition of 10-mM sodium azide. Then, worms were mounted on a microscopic glass slide. The expression of hsp-16.2 was examined by observing the fluorescence at the anterior part from the back of the pharynx as described previously (15,18).

Quantification of sod-3 Expression
L1 larvae of CF1553 transgenic worms, which carry sod-3 promoter regions fused with a GFP reporter, were treated with different concentrations of VVE extract in S-medium at 20 • C for 72 h. Then, worms were submitted to fluorescence microscopy as described previously (15,18).

Quantification of gst-4 Expression
L1 larvae of CL2166 transgenic worms, which carry gst-4 promoter regions fused with a GFP reporter, were treated with different concentrations of VVE extract in S-medium at 20 • C for 48 h. Next, the worms were induced by exposing a nonlethal dose of 20-µM juglone for 24 h. Fluorescence images were taken by fluorescence microscopy as described previously (15,18).

Determination of Subcellular Localization of DAF-16
L1 larvae of TJ356 transgenic worms were treated with different concentrations of VVE extract in S-medium for 48 h and submitted to fluorescence microscopy as described previously (15,18).

Assessment of Autofluorescent Pigment
BA17 transgenic worms were used for measuring the accumulation level of the autofluorescent pigment lipofuscin. L1 larvae of BA17 transgenic worms were treated with different concentrations of VVE extract in S-medium and maintained at 25 • C to prevent egg-laying. The media was changed every second day. On day 16, the worms were anesthetized by the addition of 10-mM sodium azide, mounted on a glass slide, and photographed. Worms were photographed using a BIOREVO BZ-9000 fluorescence microscope (λex 360/20 nm, λem 460/38 nm) as described previously (15).

Longevity Assay
The wild-type (N2) worms were used for the lifespan assay under normal conditions. Synchronization and treatment were conducted as described previously (15). In brief, N2 worms were synchronized at the L4 larval stage on NGM agar plates supplemented with VVE extracts and E. coli OP50 at 20 • C. The treatment plate was prepared by mixing VVE extracts (final concentration 50 µg/ml) with E. coli OP50 and adding on NGM agar plate overnight before use. The worms were counted during the transfer to fresh medium every day. After that, the percentages of surviving worms were documented. Worms that failed to respond to a gentle touch with a platinum wire were scored as dead and excluded from the plates. Internal hatched progeny worms were scored as censors and discarded from the assay.

Brood Size and Body Length Assay
To analyze the potential toxic effect of the extract on the reproductive system, brood size was measured as described in our previous work (15,18)

Statistical Analysis
In these studies, the results are presented as the mean ± SEM and were analyzed with GraphPad Prism 8. The experiments were performed in at least triplicate. One-way analysis of variance (ANOVA) following Bonferroni's method (post hoc) analyzed a comparison between the control and treatments. Differences were considered significant at the P ≤ 0.05 level.

Phytochemical Constituents of V. vinifera Ethanol Extract
In this study, LC-MS and HPLC were carried out for the tentative identification of the phytoconstituents in the VVE extract. A phytochemical profile is shown in Figure 1. The detected and identified compounds are listed in Supplementary Table 1 with the corresponding retention and MS/MS fragmentation data.
We tentatively identified the main compounds in the VVE extract, including resveratrol, gallic acid, apigenin, catechin, quercetin, and tannin. Fingerprinting analysis of VVE extracts using HPLC showed the presence of the bioactive compound gallic acid (18.26 mg/100 g of crude extract), catechin (55.10 mg/100 g of crude extract), epicatechin (14.22 mg/100 g of crude extract), and quercetin (197.73 mg/100 g of crude extract) (Supplementary Figure 1B, Table 2). Our results thus agree with the published chemical composition of V. vinifera leaf extracts (6,10).

Effect of V. vinifera Ethanol Extract on Glutamate-Induced Cytotoxicity in HT22 Cells
Excessive glutamate induced oxidative stress leading to neurotoxicity and neuronal cell death (2). The immortalized mouse hippocampal HT22 cells are common cell models to evaluate glutamate toxicity caused by oxidative stress. These cells lack ionotropic glutamate receptors, which exclude excitotoxicity as a cause of glutamate-triggered cell death (2).
To investigate whether the VVE extract could prevent cell death induced by glutamate, the protective effects against glutamate-induced oxidative toxicity were explored in HT22 cells using MTT, LDH assay, and cell morphological examination. First, we determined the non-cytotoxic concentration of the extract and the optimum condition of glutamate in HT22 cells. We found that the VVE extract was relatively non-cytotoxic at the tested doses (10-100 µg/ml VVE, 48 h) (Figure 2a), and the cell viability was reduced by approximately 50% at the tested doses (5-mM glutamate, 18 h) (53.9 ± 0.6% (p < 0.001) (Figure 2b).
Both antioxidant properties of VVE extract in vitro and in cells suggest that VVE extract protects against glutamate-induced cytotoxicity by inhibiting the accumulation of intracellular ROS. Previous research has indicated that an antioxidant, such as phenolic and flavonoids, strongly prevented ROS-induced neuronal cell death (19). Neuroprotective properties of resveratrol (20,21), gallic acid (22), apigenin (21), catechin (23), and quercetin (24) were also highlighted in several recent studies. Our results agreed with literature data indicating that the phenolic compounds (resveratrol, gallic acid, apigenin, catechin, quercetin, and tannin) in VVE extract may mediate antioxidant activity and neuroprotective effects in HT22 cells.

Effect of V. vinifera Ethanol Extract on Gene Expression of Antioxidant Enzymes in HT22 Cells
The antioxidant and phase II enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST), have been known as a central role of ROS-mediated cellular damage prevention (3). To further examine the mechanism of antioxidant-mediated neuroprotective effects of the VVE extract, we examined the effects of the VVE extract on antioxidant enzyme (SOD, CAT, GPx, and GST) gene expression. We found that VVE extract (50 µg/ml) significantly upregulated the expression of endogenous antioxidant enzymes, including SOD1, CAT, GPx, GSTo1, and GSTa2 (Figure 3b).
Our results are in agreement with other studies where grape leaf extracts (V. vinifera) were found to protect against oxidative damage by promoting antioxidant gene response in several models, including neuronal cells (25), C. elegans (26), and rodents (25,27,28). In accordance with previous studies, the bioactive compounds in grape leaf extracts such as resveratrol (29), catechin (29), gallic acid (22), and quercetin (30) also increased antioxidant gene expression.
In the brain, an imbalance of ROS homeostasis is involved in the pathogenesis of several neurodegenerative events (31). Antioxidant balance inside the cells requires intrinsic (endogenous enzymes) and extrinsic (dietary supplements) antioxidants for neutralizing ROS. Natural plants with antioxidant properties have been recognized as precious sources for drug discovery in age-related diseases (24,(32)(33)(34)(35). The current results demonstrated that the protective effect of VVE extract against glutamate-induced cytotoxicity is not only through suppressing intracellular ROS production but also through enhancing endogenous antioxidant and phase II enzymes in neuronal HT22 cells.

Effect of V. vinifera Ethanol Extract on Juglone-Induced Oxidative Stress in C. elegans
C. elegans is a valuable model for aging research in studying genetic and pharmacological influences of ROS (36,37). To further elucidate the antioxidant activities of the VVE extract in vivo, the oxidative resistance properties of the VVE extract were . Cells were treated with different concentrations of VVE extracts for 48 h and exposed to 5-mM glutamate for 18 h. Then, cell viability was measured by MTT (c) and LDH (d) assay. Cell morphology was observed under a microscope at 5× magnification (e). Samples were exposed to 5-mM glutamate (g) to induce toxicity. All data are shown as mean ± SEM of at least three independent experiments. #### p < 0.0001 vs. DMSO control; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with glutamate-treated cells by one-way ANOVA following Bonferroni's method (post hoc).
conducted in a C. elegans model. We first determined the survival of nematodes under oxidative stress conditions. Treatment with different concentrations of VVE extract (25-100 µg/ml) caused no significant changes in the survival rate of wild-type worms compared with the DMSO control (Supplementary Figure 2). However, under oxidative stress conditions (80-µM juglone for 24 h), the survival rate of the wildtype worms pretreated with the VVE extract was significantly increased when compared with the DMSO control (21.1 ± 1.9%) (Figure 4a) [25,50, and 100 µg/ml VVE reduced mortality by 32.8 ± 1.5% (p < 0.01), 31.5 ± 1.8% (p < 0.01) and 33.6 ± 1.2% (p < 0.001), respectively]. Stress resistance properties are closely related to antioxidant activity (31). Although VVE extracts improved the survival rate of the wild-type worms, compared with control, the survival rate did not improve in a similar range as the epigallocatechin gallate, which is a powerful antioxidant in green tea (38). Similarly, VVE extract exhibited lower scavenging activity than epigallocatechin gallate (Supplementary Figure 2). . Samples were pretreatment with VVE extracts for 48 h and exposed to 5-mM glutamate (G5) for 12 h to induce oxidative stress. Representative fluorescence micrographs of cells stained with H 2 DCFDA were observed under a fluorescence microscope (c). All data are shown as mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with glutamate-treated cells; ### p < 0.001, compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc). FIGURE 4 | Protective effect of VVE extracts against juglone-induced oxidative stress in C. elegans. VVE extracts protect against oxidative stress in wild-type C. elegans. Survival rate of wild-type (N2) worms was significantly enhanced after pretreatment with extracts (a). VVE extracts treatment reduced ROS levels in N2 worms when compared with DMSO control (b). Representative pictures of DCFDA fluorescence in wild-type (N2) worms treated with 25 µg/ml VVE (c1); 50 µg/ml VVE (c2); 100 µg/ml VVE (c3); and DMSO control (c4). In survival assay, samples were exposed to 80-µM juglone (J) to induce oxidative stress. All data are shown as mean ± SEM of at least three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc).
These results suggest that the antioxidant activity of VVE extract might be partially attributed to improving the survival rate.
However, we found that the lower concentrations of VVE extract (<10 µg/ml in HT22 cells and 25 µg/ml in worms) neither decreased intracellular ROS accumulation level nor increased survival rate under oxidative stress condition compared with the DMSO control (Supplementary Figure 4). These results suggest that the VVE extracts at moderate concentrations have antioxidant activities. All data are shown as mean ± SEM of at least three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared with DMSO control by one-way ANOVA following Bonferroni's method (post hoc).
Previous works have reported that grape seed and skin extracts have antioxidants (26) and lifespan-extending effects in C. elegans (9). The data support our assumption that the VVE leaf extract contains polyphenols (resveratrol, gallic acid, apigenin, catechin, quercetin, and tannin), which have protective effects against oxidative stress to reduce endogenous ROS levels in C. elegans.

Effect of V. vinifera Ethanol Extract on Stress Resistance Properties Mediated by the DAF-16/FoxO Pathway in C. elegans
DAF-16, the C. elegans homolog of the mammalian FOXO transcription factor, is the main transcription factor involved in stress response, metabolism, and longevity (39). DAF-16/FoxO remains inactive in the cytosol under normal conditions. In contrast, stress or specific ligands can stimulate its translocation to the nucleus, influencing stress response genes expression such as hsp-16.2, sod-3, and gst-4 (39).
To further investigate the effects of VVE extract that mediates antioxidant activity through the DAF-16/FoxO pathway, the transgenic CF1038 worms, which are the DAF-16 loss-offunction mutant, were used in survival (Figure 5a) and intracellular ROS accumulation assay (Figure 5b). Interestingly, VVE extract failed to increase the survival rate under oxidative stress (Figure 5a) and attenuate intracellular ROS levels (Figure 5b) in CF1038 worms. The data indicate that VVE extract has antioxidant activity and stress resistance in C. elegans through the DAF-16/FoxO pathway.  (Figure 6a).
The data indicate that VVE extract exhibited antioxidant properties, not only by suppressing intracellular ROS but, additionally, by modulation of the expression of stress-response genes in C. elegans, such as hsp-16.2, sod-3, and gst-4. These abilities were similar to the effects of resveratrol (41), gallic acid (15,17), catechin (32,35), and quercetin (15,24,42) on oxidative stress resistance in C. elegans via the transcription factor DAF-16/FoxO. Taken together, the results of this study strongly suggest that the VVE extract mediated antioxidant activity and stress resistance in C. elegans via the DAF-16/FoxO pathway. However, further studies are required to clarify the underlying mechanisms of the VVE extract on the neuroprotective effect in C. elegans.

Effect of V. vinifera Ethanol Extract in Aging
C. elegans is a popular model of aging and longevity (36). Several polyphenols have been reported as antiaging agents in C. elegans, such as resveratrol (43), anthocyanin (33), and quercetin (24). To examine the possible influence of VVE extract on aging, the autofluorescent pigment (lipofuscin) accumulation and lifespan were measured. The accumulation of intestinal autofluorescence (lipofuscin) in C. elegans during aging is often used as a marker of health or aging (44). We found that the VVE extract significantly decreased the level of lipofuscin accumulation in late adult worms (16 days) (Figure 7a).
Despite the antioxidant capacity in vitro and in vivo and aging marker reduction, VVE extract did not show any lifespanprolonging effects in wild-type worms in normal conditions (Figure 7b). These abilities were similar to the effects of resveratrol in the life span of C. elegans under normal conditions (43). However, the resveratrol show strongly increased life span effects in C. elegans under conditions of oxidative stress (43). Possibly, the antiaging effects of VVE extracts are linked to antioxidant effects. The effects of VVE extract on the life span of C. elegans under oxidative stress conditions will be an interesting topic for future study.
To exclude the toxic effect on the reproductive system and dietary restriction system induced by VVE extract, we further measured brood size and body length. Brood size (Figure 7c) and body length (Figure 7d) in wild-type worms were not affected by different concentrations of VVE extract. These data indicated that the effects of VVE extract did not interfere with the fertility rate nor with body development (e.g., via dietary restriction) as mentioned in the literature as toxicity markers (33).

CONCLUSION
Oxidative stress has been connected to neurodegenerative diseases (1,2). In this study, HT22 hippocampal neuronal cells and C. elegans models were used to study the protective effects of VVE extract against oxidative stress as in vitro and in vivo studies. We found that the VVE extract protects against glutamate-induced oxidative toxicity in HT22 hippocampal neuronal cells and against juglone-induced oxidative stress in C. elegans. The neuroprotective action of the VVE extract in hippocampal neuronal (HT22) cells is mediated via inhibition of ROS accumulation and enhancing endogenous antioxidant enzymes. In addition, the VVE extract exhibits oxidative resistance properties in C. elegans involved in the DAF-16/FoxO signaling pathway. VVE reduced age-related markers (lipofuscin), although it did not extend the life span of C. elegans under normal conditions. These studies first report the phytochemical constituents and antioxidant properties of V.vinifera leaf extract. The leaf extract might be considered as an alternative supplement or medicine to defend against oxidative stress and neurodegenerative diseases. In vivo intervention studies with more complex model organisms are required to support the therapeutic potential of the VVE extract for agerelated neurodegenerative disorders.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
CD performed the experiments, analyzed data, and was a major contributor in writing the manuscript. PR performed the gene expression assay by RT-PCR. CD, PR, SZ, and XG designed the study and prepared media and reagents. MW review and editing the manuscript. MW and TT provided materials for the study and conceived and supervised research. MW, TT, PR, SZ, and XG corrected the manuscript. All authors contributed to the article and approved the submitted version.